1 1 The role of phospholipids in nutrition and metabolism of teleost fish 2 3 4 Douglas R. Tocher 1* , Eldar Å. Bendiksen 2 , Patrick J. Campbell 2 and J. Gordon 5 Bell 1 6 7 8 1 Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland, UK. 9 2 BioMar AS, Nordregt. 11, N-7484 Trondheim, Norway. 10 11 12 13 14 Keywords: Teleosts; fish; phospholipids; requirements; nutrition; metabolism; dietary supply; 15 plant meals; cholesterol 16 17 18 19 20 21 22 23 *, Correspondence to: Douglas Tocher, Institute of Aquaculture, University of Stirling, Stirling 24 FK9 4LA, United Kingdom. Tel. No: +44 1786 467996; Fax No: +44 1786 472133; Email: 25 [email protected]26 27
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1
1
The role of phospholipids in nutrition and metabolism of teleost fish 2
3
4
Douglas R. Tocher1*, Eldar Å. Bendiksen2, Patrick J. Campbell2 and J. Gordon 5
Bell1 6
7
8
1Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland, UK. 9
It has been known for almost 25 years now that inclusion of intact phospholipids in the diet could 28
improve culture performance of various freshwater and marine fish species. The primary 29
beneficial effect was improved growth in both larvae and early juveniles, but also increased 30
survival rates and decreased incidence of malformation in larvae, and perhaps increased stress 31
resistance. Determination of absolute dietary requirements has been hampered by the use, in 32
different dietary trials, of a wide range of phospholipid preparations that can vary greatly both in 33
phospholipid content and class composition. Larval studies have been compromised further by the 34
need on many occasions to supply phospholipid through enrichment of live feeds with subsequent 35
re-modelling of the phospholipid and fatty acid compositions. Generally, the levels of 36
phospholipid requirement are around 2 - 4% of diet for juvenile fish and probably higher in larval 37
fish. The effects were restricted to young fish, as a requirement for dietary phospholipids has not 38
been established for adult fish, although this has been virtually unstudied. As the majority of 39
studies have used crude mixed phospholipid preparations, particularly soybean lecithin, but also 40
other plant phospholipids and egg yolk lecithin, that are enriched in several phospholipids, it has 41
been difficult to elucidate which specific phospholipid classes impart beneficial effects. Based on 42
the few studies where single pure phospholipid species have been used, the rank order for efficacy 43
appears to be phosphatidylcholine > phosphatidylinositol > phosphatidylethanolamine > 44
phosphatidylserine. The efficacy of other phospholipid classes or sphingolipids is not known. The 45
mechanism underpinning the role of the phospholipids in larval and early juvenile fish must also 46
explain their lack of effect in adult fish. The role of phospholipids appears to be independent of 47
fatty acid requirements although the presence of an unsaturated fatty acid at the sn-2 position may 48
be important. Similarly, the phospholipid requirement is not related to the delivery of other 49
essential dietary components such as the bases choline and inositol. Studies also suggested that the 50
phospholipid effect was not due to generally enhanced emulsification and digestion of lipids. 51
Rather the evidence led to the hypothesis that early developing stages of fish had impaired ability 52
to transport dietary lipids away from the intestine possibly through limitations in lipoprotein 53
synthesis. The current hypothesis is that the enzymic location of the limitation is actually in 54
phospholipid biosynthesis, perhaps the production of the glycerophosphobase backbone and that 55
dietary supplementation with intact phospholipids in larvae and juvenile fish compensated for this. 56
Thus, dietary phospholipids increase the efficiency of transport of dietary fatty acids and lipids 57
from the gut to the rest of the body possibly through enhanced lipoprotein synthesis. 58
59
3
Contents 59 60
1. Introduction 61
2. Roles of phospholipids 62
2.1 Structural 63
2.2 Regulation of metabolism and physiology 64
2.3 Eicosanoids 65
2.4 Phosphoinositides 66
2.5 Platelet-activating factor 67
2.6 Energy provision 68
3. Phospholipid biosynthesis 69
3.1 De novo synthesis 70
3.2 Turnover and remodelling 71
4. Phospholipid digestion, absorption and transport 72
4.1 Digestion 73
4.2 Absorption 74
4.3 Transport 75
5. Phospholipid requirements in fish 76
5.1 Early studies 77
5.2 Limitations in studies 78
5.3 Requirement criteria 79
5.4 Range of species and developmental stages 80
5.5 Quantitative requirements 81
5.6 Qualitative requirements 82
6. Mechanisms of requirement for intact phospholipids in larvae and juveniles 83
6.1 Improved diet qualities 84
6.2 Improved digestion 85
6.3 Provision of essential components 86
6.4 Fatty acids (energy and essential fatty acids) 87
6.5 Phosphorous 88
6.6 Choline and inositol 89
6.7 Provision of intact phospholipid 90
7. Dietary phospholipids and sustainability 91
4
7.1 Sustainability 92
7.2 Dietary sources of phospholipids 93
7.2.1 Oils 94
7.2.2 Meals 95
7.3 Analysis of phospholipid content and composition in fish diets and ingredients 96
7.4 Dietary cholesterol 97
8. Conclusions 98
References 99
100 101
5
1. Introduction 101
102
Phospholipid is a general term that includes all lipids containing phosphorus. However, it is a 103
term often mistakenly equated with phosphoglycerides, the most common of the phospholipids. 104
Phosphoglycerides are characterised by a common backbone of phosphatidic acid (PA), formed 105
from L-glycerol 3-phosphate with two fatty acids esterified on positions 1 and 2. The major 106
phosphoglycerides of animal including fish tissues phosphatidylcholine (PC), 107
phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI), 108
respectively, are formed by the esterification of the “bases” choline, ethanolamine, serine and 109
inositol to the phosphate group of PA. A common generalisation has been that saturated and 110
monounsaturated fatty acids are preferentially esterified on position sn-1 of phosphoglycerides 111
with polyunsaturated fatty acids (PUFA) preferentially esterified on position sn-2. However, 112
there are many exceptions to this, such as the di-docosahexaenoyl phosphoglycerides that are 113
abundant in the retina of fish, specifically in rod outer membrane segments (Bell and Tocher, 114
1989; Bell and Dick, 1991). Another phosphorus-containing polar lipid is the sphingolipid, 115
sphingomyelin, a complex lipid based on the long-chain amino alcohol sphingosine. All 116
sphingolipids contain a long chain, generally saturated or monounsaturated fatty acid, linked to 117
the amino group of sphingosine (forming a ceramide), with different polar groups, such as, 118
phosphocholine in the case of sphingomyelin, attached to the primary alcohol group. 119
For the remainder of this review we will exclusively use the term “phospholipid” when, in 120
actual fact, phosphoglyceride would be the more correct term, as described above. However, the 121
use of phospholipid will be consistent with the vast majority of studies that have been reported in 122
the literature. Similarly, the abbreviations PC, PE, PS, PI and PA, as used above, are not strictly 123
correct but are by far the most commonly used for the major phosphoglyceride classes and so 124
will be used throughout this review. 125
126
2. Roles of phospholipids 127
128
2.1 Structural 129
The amphipathic structure of phospholipids, having both hydrophilic (sn-3 phosphate/head-130
group) and hydrophobic (sn-1 and -2 fatty acids) regions means they have the same central role in 131
the structure of cell membrane bilayers in fish as they do in mammals. Although not well studied 132
in fish, there is evidence that the asymmetric distribution of phospholipids in cell membranes 133
6
established in mammals, with choline-containing phospholipids, PC and sphingomyelin 134
concentrated in the outer leaflet and PE, PS and, to a lesser extent, PI concentrated in the inner 135
leaflet, is also a feature of fish cell membranes (Kagan et al., 1984). The dynamic changes in the 136
composition and metabolism of the phospholipids in biomembranes in response to environmental 137
factors, especially temperature, have been reviewed previously (Hazel and Williams, 1990; 138
Hochachka and Mommsen, 1995). Their amphipathicity is also central to the role phospholipids 139
have in the structure of the lipoproteins that are important in the extracellular transport of lipids 140
in the blood and lymph. The lipoproteins are described in more detail in section 4.3 but, 141
essentially, phospholipids enable hydrophobic lipids such as triacylglycerols and steryl esters to 142
be transported in aqueous environments by forming, along with cholesterol and proteins, the 143
lipid/water interfaces (Tocher, 1985). It can be argued that phospholipids also have an important 144
structural role in the digestion of lipids as they are essential in forming intra-luminal mixed 145
micelles along with bile salts and dietary lipids (see section 4.2) (Olsen and Ringø, 1997). This 146
phospholipid is not entirely of dietary origin as fish bile can contain variable amounts of 147
phospholipid (Moschetta et al., 2005). It is thought that biliary phospholipid has two roles in the 148
bile as the formation of mixed micelles with bile salts not only solubilises biliary cholesterol, but 149
also has a cytoprotective effect, protecting biliary tract epithelium from the cytotoxic effects of 150
bile salts (Moschetta et al., 2005). PC is usually the major phospholipid in fish bile but 151
sphingomyelin can also be a major component, particularly in species with lower biliary 152
phospholipid levels and phospholipid:bile salt ratios (Moschetta et al., 2005). 153
154
2.2 Regulation of metabolism and physiology 155
Phospholipids are important precursors for a range of highly biologically active mediators of 156
metabolism and physiology including eicosanoids, diacylglycerol (DAG), inositol phosphates 157
and platelet activating factors (PAFs). With the possible exception of eicosanoids, these areas of 158
phospholipid metabolism are relatively poorly studied in fish. However, there is evidence that all 159
these pathways occur in fish and that the phospholipid-derived mediators play similar roles in 160
fish as they do in mammals (Tocher, 1995, 2003). 161
162
2.2.1 Eicosanoids 163
Phospholipids are the source of the substrate fatty acids for the formation of eicosanoids, a range 164
of highly bioactive derivatives of, in particular C20 highly unsaturated fatty acids (HUFA), 165
7
especially arachidonic acid (ARA, 20:4n-6) and eicosapentaenoic acid (EPA, 20:5n-3). Fatty 166
acids released from membrane phospholipids by the action of phospholipase A2 are converted by 167
either cyclooxygenase enzymes, which produces cyclic oxygenated derivatives, collectively 168
called prostanoids, including prostaglandins, prostacyclins and thromboxanes, or lipoxygenase 169
enzymes which produce linear oxygenated derivatives including hydroperoxy- and hydroxy fatty 170
acids, leukotrienes and lipoxins. Eicosanoids are produced by many tissues in response to various 171
extracellular stimuli and are autocrines, hormone-like compounds of short half-life that act in the 172
immediate vicinity of their production. They are implicated in many physiological processes 173
including blood clotting and cardiovascular tone, immune and inflammatory responses, 174
reproduction, renal and neural functions. The distribution and production of eicosanoids in fish 175
species and tissues and their possible roles have been reviewed previously (Sargent et al., 1989, 176
2002; Tocher, 1995, 2003). 177
178
2.2.2 Phosphoinositides 179
Phosphorylated derivatives of PI (e.g. PIP2) are produced by the action of various kinases and 180
phosphatases on the inositol ring. The phosphorylated derivatives of PI may themselves have 181
important roles in metabolism including golgi/lysosome/endosome trafficking, cytoskeleton 182
regulation and cell survival. The most studied pathway though is the cleavage of PIP2 by 183
phospholipase C-β to produce two intracellular second messengers, DAG, and the inositol 184
phosphate, IP3, in response to various hormones and effectors (Berridge, 1987). IP3 induces 185
calcium mobilisation from the endoplasmic reticulum, and increased intracellular Ca2+ and DAG 186
are activators of protein kinase C, a threonine/serine kinase and important regulator of 187
metabolism (Kuo et al., 1980). DAG can also be phosphorylated by DAG kinase to form PA and 188
lyso-PA, increasingly being recognised as other important intracellular regulators of a growing 189
number of signalling proteins (Hao et al., 2007). Other phospholipids, including PC and PS, also 190
have important metabolic functions in mammals as specific activators of protein kinase C, along 191
with DAG and Ca2+ ions (Kuo et al., 1980). Protein kinase C and PS-activation has been reported 192
in rainbow trout (Oncorhynchus mykiss) spleen and dogfish (Scyliorhinus canicula) rectal gland 193
(Bell and Sargent, 1987), and a role for protein kinase C has been implicated in the stimulation of 194
steroidogenesis in goldfish (Carassius auratus) gonadal tissues (Van Der Kraak, 1990; Wade and 195
Van Der Kraak, 1991). Inositol phospholipid metabolism including the roles of phospholipase C 196
8
and Li-sensitive phosphatases have been studied in metabolically active electrocytes from the 197
electric ray (Discopyge tschudii) (Arias and Barrantes, 1990). 198
199
2.2.3 Platelet-activating factor (PAF) 200
PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, is a biologically active phospholipid 201
synthesised by inflammatory cells and a potent mediator of many leukocyte functions, including 202
platelet aggregation, inflammation, and anaphylaxis (Snyder, 1987). PAF is biosynthesized from 203
lyso-PAF and acetyl CoA by the enzyme lyso-PAF acetyltransferase and degraded (thereby 204
terminating activity) by a group of enzymes called PAF acetylhydrolases that are related to 205
phospholipase A2. In mammals, the reacylation of lyso-PAF is highly specific for ARA, and it is 206
the 1-alky-2-arachidonyl-glycerophosphocholine species that is the substrate for the synthesis of 207
PAF via an ARA-specific phospholipase A2 that also produces ARA for eicosanoid synthesis 208
(Snyder, 1987). Synthesis of PAF has been demonstrated in rainbow trout (Turner and Lumb, 209
1989) and PAF acetylhydrolase activity has been reported in fish serum (Cabot et al., 1984). 210
211
2.3 Energy production 212
Any lipid class containing fatty acids can act as a source of energy, which is released through β-213
oxidation of the acyl chains producing acetyl-CoA and NADH that are further metabolised via 214
the tricarboxylic acid cycle and oxidative phosphorylation, respectively. Triacylglycerols are the 215
primary class for lipid storage and energy provision, but phospholipids can serve as a source of 216
energy in fish in certain circumstances, such as embryonic and early larval development. 217
Upon hatching, the larvae of many fish species have immature mouthparts and intestinal tract 218
and are unable to commence exogenous feeding immediately. Therefore, throughout 219
embryogenesis in the egg and then during early larval development up to first feeding, the larvae 220
gain nutrition from endogenous energy reserves in the yolk. Essentially there are two main types 221
of fish egg, those with oil globule(s) having relatively high levels of neutral lipids (20-50% of 222
egg total lipid), and those without an oil globule, low levels of neutral lipids (< 15% of total lipid) 223
and, consequently, high levels of phospholipids, predominantly PC (Wiegand, 1996; Salze et al., 224
2006). In eggs of the latter type, including those from Atlantic herring (Clupea harengus), cod 225
(Gadus morhua), halibut (Hippoglossus hippoglossus) and plaice (Pleuronectes platessa), 226
phospholipid, predominantly PC, is utilised during both embryogenesis and early larval 227
development (Tocher et al., 1985a; Fraser et al., 1988; Rainuzzo et al., 1992; Finn et al., 1995; 228
9
Ronnestad et al., 1995 (Finn et al., 1995; Ronnestad et al., 1995). In contrast, neutral lipid-rich 229
eggs such as those from turbot (Psetta maximus), utilise primarily triacylglycerols, but also steryl 230
and wax esters where present (Sargent et al., 1989, 2002; Rainuzzo et al., 1992; Weigand, 1996). 231
Another relatively common feature observed during development is the conservation and/or 232
synthesis of PE, as reported in both the phospholipid-rich eggs of cod (Fraser et al., 1988), plaice 233
and halibut (Rainuzzo et al., 1992; Ronnestad et al., 1995), and also the neutral lipid-rich eggs of 234
Atlantic salmon (Salmo salar) and turbot (Cowey et al., 1985; Rainuzzo et al., 1992), Senegal 235
sole (Solea senegalensis) (Mourente and Vazquez, 1996) and dentex (Dentex dentex) (Mourente 236
et al., 1999). This results in a decrease and normalisation of the PC : PE ratio as development 237
proceeds, from the high values seen in most marine fish eggs to values normally observed in fish 238
tissues. This is particularly the case in the phospholipid - rich eggs, dominated by PC, where PC 239
is catabolised during embryogenesis, but has also been observed in Atlantic salmon where PC, 240
originally accounting for over 94% of the phospholipid in the egg, was continuously metabolized 241
so that in the fry a PC : PE ratio approaching that of salmon muscle was obtained (Cowey et al., 242
1985). 243
These data show a decrease in the absolute amount of phospholipid, usually PC, during 244
embryogenesis and early larval development, particularly in phospholipid-rich marine fish eggs. 245
A possible consequence of complete catabolism of phospholipid for energy would be the loss of 246
important PUFA. However, in an early study in Atlantic herring, much of the PUFA liberated by 247
the catabolism of PC was selectively retained in the neutral lipid pool (Tocher et al., 1985b). 248
About one third of the docosahexaenoic acid (DHA, 22:6n-3) released during PC catabolism in 249
cod eggs was incorporated into neutral lipids (Fraser et al., 1988). Similarly, in halibut, over 60% 250
of the DHA from PC hydrolysis was selectively retained in PE and neutral lipids (Ronnestad et 251
al., 1995). However, DHA was also a quantitatively important fuel in halibut eggs with almost 252
40% of the DHA being catabolised (Ronnestad et al., 1995). Similarly, in cod the fatty acids in 253
PC were catabolised non-selectively with both EPA and DHA being utilised (Finn et al., 1995). 254
255
3. Phospholipid biosynthesis 256
257
3.1 De novo synthesis 258
In mammals, phospholipids can be synthesised de novo by two main mechanisms (see Lykidis, 259
2007). One utilizes a cytidine diphosphate (CDP)-activated polar head group for attachment to 260
1,2-DAG, and the other utilizes CDP-activated DAG and an inactivated polar head group (Fig.1). 261
10
They both initially proceed by esterification of glycerol-3-phosphate with two activated fatty 262
acids (acyl-CoA) to form PA. Action of PA phosphatase on PA results in the production of 263
DAG, which can then react with CDP-choline or CDP-ethanolamine to produce PC and PE, 264
respectively. Alternatively, CTP reacts with PA to form CDP-DAG, which can then combine 265
with serine and inositol to form PS and PI, respectively. Reaction of CDP-DAG with glycerol-3-266
phosphate leads to phosphatidylglycerol (PG) and combination of PG with a further CDP-DAG 267
leads to the formation of diphosphatidylglycerols that include cardiolipin. PS can also be formed 268
in the membrane bilayer by exchange reactions of serine for ethanolamine or choline in PE and 269
PC, respectively. PS can be converted back to PE by decarboxylation and PC can also be formed 270
by three successive methylations of PE utilizing S-adenosylmethionine. 271
The pathways of de novo phospholipid biosynthesis have not been extensively studied in fish, 272
but all the necessary phospholipid biosynthetic enzymes and protein families can be found in the 273
fugu (Takifugu rubripes) and zebrafish (Danio rerio) genomes (Lykidis, 2007). The existing 274
biochemical evidence also suggests that pathways are essentially the same as in mammals 275
(Tocher, 1995). Incubation of trout liver microsomes with 14C-glycerol-3-phosphate in the 276
presence of palmitoyl-CoA, resulted in three-quarters of the radioactivity being recovered in total 277
phospholipids, predominantly PA and lyso-PA but also other phospholipid classes, demonstrating 278
glycerol-3-phosphate acyltransferase activity and supporting the conclusion that phospholipid 279
biosynthesis in fish proceeded via a PA intermediate (Holub et al., 1975a). Activity of α-280
glycerophosphate acyltransferase in fish was later confirmed, although it was reported to be 281
extremely low compared to other animals (Iritani et al., 1984). Studies on the incorporation of 282 14C-palmitate and 14C-glycerol-3-phosphate into lipids in carp (Cyprinus carpio) intestinal 283
homogenates in the presence of CTP, CDP-choline and CDP-ethanolamine showed phospholipid 284
biosynthesis from PA and DAG intermediates, indicating synthesis from moieties other than 285
lyso-phospholipids (Iijima et al., 1983). However, little is known about what determines the fate 286
of newly-synthesised DAG (PC or triacylglycerol synthesis) in fish, but it has been suggested that 287
fatty acid composition of the DAG may differentially affect the activities of the biosynthetic 288
enzymes (Oxley et al., 2007). Early studies demonstrated the presence of CDP-choline:DAG 289
choline phosphotransferase activity in trout liver microsomes (Holub et al., 1975b), and brain and 290
liver from goldfish (Leslie and Buckley, 1975), establishing that the choline phosphotransferase 291
(CPT) pathway for the biosynthesis of PC operated in fish. Similarly, synthesis of PE from DAG 292
and the presence of CDP-ethanolamine phosphotransferase (i.e. the EPT pathway) were 293
subsequently demonstrated in trout hepatocytes (Hazel, 1990). The activity of CTP in 294
11
microsomes from intestinal mucosa in adult Atlantic salmon (~ 1 kg) was comparable to the 295
reported mammalian activity (Oxley et al., 2005). Thus, synthesis via DAG and CPT/EPT was 296
confirmed as the predominant pathway for phospholipid synthesis in fish as in mammals (Hazel, 297
1990). Although not directly studied or demonstrated in fish, the presence in fish of PI, PG and 298
cardiolipin, that cannot be synthesised via the phosphotransferase pathways, implies the presence 299
of the alternative CDP-DAG pathway in fish. Similarly, not all the phospholipid inter-conversion 300
pathways have been studied in fish, but the activities of PE-methyltransferase (PE to PC) and PS-301
decarboxylase (PS to PE) have been demonstrated in trout hepatocytes (Hazel, 1990). 302
303
3.2 Phospholipid turnover and remodelling 304
Remodelling and turnover of phospholipids can involve both head groups and fatty acyl chains. 305
The mechanisms whereby phospholipid classes (PC, PE, PS and PI, etc.) could be inter-converted 306
are described above (see Fig.1), and phospholipid remodelling at this level is certainly involved 307
in homeoviscous adaptation of biological membranes, especially in relation to temperature 308
adaptation. Thus, cold acclimation in fish has been associated with increased proportions of PE 309
and decreased proportions of PC (Hazel and Williams, 1990). Although the activities of CDP-310
choline and CDP-ethanolamine phosphotransferase were both reduced at lower temperature, 311
synthesis of PC was more dependent upon temperature than that of PE and, in addition, PS 312
decarboxylase activity was increased (Hazel, 1990). As a result, the PC : PE synthesis ratio was 313
positively correlated with temperature and so these mechanisms may underpin the increased PE : 314
PC ratio in cold-acclimated fish (Hazel, 1990). 315
Fatty acid retailoring of phospholipids through acyl exchange reactions has been relatively 316
more studied in fish, again most often in relation to homeoviscous adaptation to environmental 317
changes. The enzymes required for deacylation of phospholipids and reacylation of lyso-318
phospholipids, and thus for the turnover of phospholipids, have been demonstrated in fish. 319
Intracellular phospholipase A activities have been reported in muscle tissue from a variety of fish 320
species including rainbow trout and cod (see Tocher, 2003), and a review of muscle lipase 321
activities indicated that hydrolysis of phospholipids in fish was primarily under the control of 322
phospholipases A1 and A2 (Shewfelt, 1981). The activity of liver microsomal phospholipase A2 323
has been studied in trout (Neas and Hazel, 1985), whereas cytosolic phospholipase A2 was 324
demonstrated to be the principal phospholipase activity during zebrafish embryogenesis (Farber 325
et al., 1999). AcylCoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase activity has also 326
12
been demonstrated in trout liver microsomes (Holub et al., 1976). It is presumed that the fatty 327
acyl and head group specificities of the enzymes involved in the deacylation/reacylation turnover 328
processes have important roles in maintaining the characteristic fatty acyl distribution among the 329
phospholipid classes (Tocher, 2003). Thus, PC is characterized by having high 16:0 (higher than 330
any other class) and relatively lower PUFA (i.e. lower than PE and PS); PE generally displays 331
intermediate levels of saturated and monounsaturated fatty acids and high levels of PUFA which 332
are relatively evenly split between C20 and C22 PUFA; PS is characterized by high 18:0 and high 333
PUFA, predominantly C22 PUFA; PI also has high 18:0, but relatively lower PUFA, which is 334
predominantly C20, particularly ARA, and a low n-3 to n-6 ratio (Tocher, 1995). 335
336
4. Phospholipid digestion, absorption and transport 337
338
4.1 Digestion 339
Digestion of dietary phospholipids is relatively unstudied in fish, but it is presumed that the 340
mechanisms are generally similar to those in mammals whereby phospholipids are digested by 341
intestinal phospholipase A2, secreted by the pancreas, resulting in the formation of 1-acyl lyso-342
phospholipids and free fatty acids that are absorbed by the intestinal mucosal cells (Henderson 343
and Tocher, 1987; Sargent et al., 1989). Lyso-phospholipids have a detergent action and so aid 344
the digestion of other lipids and, indeed, some phospholipid is secreted in the bile as substrate for 345
phospholipase A2. The lack of a discrete pancreas in most teleost species has hampered studies 346
on intestinal lipolysis in fish in general and, although several studies have demonstrated 347
phospholipase A2 activities in intestinal tissue of fish, it has been difficult to establish 348
conclusively if they were intracellular activities or secreted (see Tocher, 2003). However, two 349
phospholipase A2 isoforms were purified from red sea bream hepatopancreas and 350
characterisation confirmed them as low molecular weight, Ca2+-dependent group I (secretory) 351
forms (Ono and Iijima, 1998). Phospholipase A2 activity was only detected several days after 352
mouth opening in marine fish larvae (Zambonino-Infante and Cahu, 2001). There are few data 353
on rates of phospholipid hydrolysis, but digesta from turbot rectum was shown to hydrolyse PC, 354
although at slower rates than triacylglycerol and sterol esters (Koven et al., 1994). 355
356
4.2 Absorption 357
13
The mechanisms of absorption of the products of phospholipid digestion have not been 358
extensively studied in fish but are assumed to be generally similar to that in mammals. Thus, the 359
hydrolytic products, 1-acyl lyso-phospholipids and free fatty acids, will be associated with all the 360
other products of fat digestion in mixed micelles with bile salts, that diffuse to the intestinal 361
mucosa where uptake into the enterocytes occurs, probably mainly by passive diffusion. 362
The concentration of lyso-phospholipids is very low in fish plasma and so it is assumed 363
that in the intestinal mucosa the majority of the 1-acyl lyso-phospholipids are re-esterified with 364
activated free fatty acids in the microsomes before export from the enterocytes into the 365
circulatory system (Sargent et al., 1989). This is supported by a study measuring the absorption 366
of 14C-dioleoyl PC force-fed to carp (Iijima et al., 1990). Radioactivity in plasma lipoproteins 367
20 - 28 h after force-feeding was associated with various lipid classes including PC, and the 368
radioactivity associated with the sn-1 position was more than twice that associated with the sn-2 369
position (Iijima et al., 1990). There appears to be some specificity in the reacylation processes as 370
a study of fatty acid uptake by isolated enterocytes from rainbow trout showed that the recovery 371
of HUFA and 16:0 in phospholipids was higher than that of other fatty acids (Perez et al., 1999). 372
373
4.3 Transport 374
Phospholipids are transported in the blood of most fish as lipoproteins as they are in mammals 375
(see Tocher, 1995). Lipoproteins include chylomicrons, very-low density lipoproteins (VLDL), 376
low density lipoprotein (LDL) and high density lipoprotein (HDL) containing 8, 21, 25 and 29 % 377
of total weight as phospholipids, respectively (Chapman et al., 1978). However, as a percentage 378
of the total lipids, the proportion of phospholipids in trout lipoproteins ranges from around 9% in 379
chylomicrons to 23 % in VLDL, 35 % in LDL and 53 % in HDL (Chapman et al., 1978), 380
although carp lipoproteins contained proportionally more phospholipid than trout, salmon or 381
sardine (Sardinops) lipoproteins with the total lipid from VLDL, LDL and HDL containing 30 %, 382
58 % and 82 % phospholipid, respectively (Iijima et al., 1989). The relative proportions of the 383
plasma lipoproteins in fish are dependent upon age, nutrition and reproductive cycle and also 384
vary with species, but is a constant characteristic of each species depending upon dietary status 385
(Fremont and Leger, 1981). 386
The re-esterification reactions in the intestine occur primarily in the endoplasmic reticulum 387
leading to the production of chylomicrons and, to a lesser extent, VLDL (see Tocher, 1995). 388
Transport from the intestine to the liver is primarily via the lymphatic system as in trout and 389
14
tench (Tinca tinca) although, in carp, intestinal lipoproteins may be transported directly to the 390
liver via the portal system (Noaillac-Depeyre and Gas, 1974; Sire et al., 1981). Although the 391
effects of phospholipid content and composition on the production of intestinal lipoproteins in 392
fish have not been studied, as chylomicrons and VLDL have very different phospholipid 393
contents, variable proportions of dietary phospholipid could be accommodated by varying the 394
relative proportions of the intestinal lipoproteins produced. In common with most vertebrates, PC 395
appears to be the predominant phospholipid class in fish lipoproteins, although the precise 396
phospholipid class composition in fish plasma lipoproteins has been rarely reported (Nelson and 397
Shore, 1974; Chapman, 1980). 398
The processes of lipoprotein metabolism and remodelling are essentially the same in fish as in 399
mammals and are described in detail previously (Tocher, 1995, 2003) and so are only 400
summarised here. Although some may be produced in the intestine, the majority of VLDL is 401
synthesized in the liver. Triacylglycerols in chylomicrons and VLDL are hydrolyzed by 402
lipoprotein lipase (LPL) and hepatic lipase (HL) at tissue sites with the hydrolysis products being 403
absorbed. Excess surface constituents, including phospholipids, dissociate as nascent HDL 404
particles, leading to the formation of intermediate density lipoprotein (IDL) and LDL. Nascent 405
HDL can take up free cholesterol from peripheral tissues which is then esterified by the action of 406
lecithin:cholesterol acyl transferase (LCAT), a plasma enzyme which catalyzes the esterification 407
of cholesterol using fatty acid from PC, resulting in the production of mature HDL. All the major 408
enzymes of lipoprotein metabolism and remodelling, including LPL, HL and LCAT, have been 409
demonstrated in the plasma of various fish (Tocher, 1995). 410
Although virtually unstudied in fish, by analogy with the system characterized in mammals, 411
phospholipids can probably be taken up into the tissues by two or three main mechanisms. In 412
liver and some other tissues, receptor-mediated endocytosis, via receptors for B/E and E 413
apoproteins, are important pathways for LDL (apo B), VLDL- and chylomicron-remnants (apo B 414
and E) and a subfraction of HDL rich in apo E. Non-specific pinocytosis may be an important 415
pathway for all lipoproteins, but particularly LDL and HDL. In liver, around 30 % of LDL uptake 416
is via a non-receptor-mediated pathway in mammals. Finally, phospholipids and other surface 417
components of VLDL and chylomicrons may be taken up or exchanged via direct interaction 418
with the endothelial cell membranes in the tissues. 419
420
5. Phospholipid requirements of fish 421
15
422
All the available evidence suggests that phospholipid metabolic pathways, including those of de 423
novo phospholipid biosynthesis, are essentially the same in fish as in mammals. However, there 424
is now substantial evidence that at least some species of fish, both freshwater and marine, may 425
have only a limited capacity to synthesise phospholipids de novo both at larval and early juvenile 426
stages (see Coutteau et al., 1997). This may reflect the fact that many fish larvae receive an 427
abundance of phospholipids in their natural diets, whether from yolk sac lipids prior to first 428
feeding or from natural prey at and after first feeding. The same situation may well hold for de 429
novo biosyntheses of cholesterol and sphingolipids, which have been scarcely studied in fish. 430
431
5.1 Early studies 432
During studies in the early 1980s investigating microparticulate diets as replacements for live 433
feeds for rearing larval marine fish, Kanazawa and coworkers observed that dietary phospholipids 434
were essential for normal growth and survival of fish larvae such as ayu (Plecoglossus altivelis) 435
and red sea bream (Pagrus major) (Kanazawa, 1985). Improved growth and survival rates were 436
obtained with 10-100 day-old larval ayu when the diets were supplemented with 3% chicken-egg 437
lecithin, bonito-egg lecithin fraction (PC) or soybean lecithin (Kanazawa et al., 1981, 1983a). 438
Bonito-egg cephalin fraction (PE) was not as effective as the PC fraction (Kanazawa et al., 1981, 439
1983a). Similar results were found with larval red sea bream (Kanazawa et al., 1983b). These 440
workers also reported that the addition of some phospholipid to the microparticulate diet 441
formulation reduced the incidence of malformations (Kanazawa et al., 1981, 1983a,b). These 442
initial reports stimulated a significant amount of further research into phospholipid requirements 443
of larvae and juveniles of a number of other fish species (see Coutteau et al., 1997). The 444
following sections summarise these studies. 445
446
5.2 Limitations in studies 447
Studies investigating phospholipid requirements in fish have some limitations based on the 448
requirement for a deficient basal diet. For juvenile fish, deficient diets can be formulated by 449
careful choice of the raw ingredients. However pelleted diets are not suitable for marine fish 450
larvae, and the traditional diets, live feeds, are unsuitable as they contain phospholipids and 451
further enrichment can be difficult (Nordgreen et al., 2007). Therefore, microdiets are required, 452
but they have been difficult to produce and were not available for all the studies (Baskerville-453
Bridges and Kling, 2000; Yufera et al., 2000; Cahu and Zambonino Infante, 2001; Koven et al., 454
16
2001; Robin and Vincent, 2003; Kvale et al., 2006). Irrespective of whether pelleted feeds or 455
microdiets are used, they can both have problems of low palatability as the formulations must be 456
either fish meal-free or use defatted fish meal. As a result the majority of studies have been 457
performed with casein-based diets and the resultant impact on attractant and palatability factors 458
can lead to acceptance problems. 459
Supplementation of phospholipids to basal deficient diets can pose problems as, although 460
commercial phospholipid preparations (lecithins) are available, they are not pure products. 461
Lecithin is, strictly speaking, the trivial name for PC, but has been used commercially to describe 462
a range of phospholipid-enriched products usually obtained either from chicken eggs or as a by-463
product of vegetable oil processing (see section 7.2.1). The commercially available lecithins can 464
vary greatly in purity, and have highly variable phospholipid contents and class compositions, as 465
well as very different fatty acid compositions. An alternative is to use pure phospholipid species 466
(classes), but these have limited availability and are costly, although it is possible to purify 467
individual phospholipid classes in the laboratory (Geurden et al., 1998a). Irrespective of the 468
specific phospholipid supplemented, replacement of one lipid class with another, i.e. 469
triacylglycerol with phospholipid, will invariably alter the fatty acid composition of the diets and 470
this can be difficult to fully control. This is also the case in comparing different phospholipid 471
preparations. Marine phospholipids were superior to soybean phospholipids in promoting growth 472
in turbot larvae, but fatty acid compositions were not controlled and so the marine phospholipid 473
diets also had far superior fatty acid profiles in terms of n-3 HUFA content (MacQueen Leifson 474
et al., 2003). 475
Finally, another major limitation over all the studies is that older fish have not been 476
investigated. The rationale presumably being that a requirement for intact phospholipids would 477
only manifest itself in fish receiving diets having very low levels of intact phospholipids. Once 478
being fed extruded diets based on fish meal, the diets will contain a luxus of intact phospholipids 479
of all classes and thus a requirement, if any, would be satisfied. 480
481
5.3 Requirement criteria 482
The criteria used to measure requirements for phospholipids have generally been gross 483
performance indicators including growth, survival and development. Thus, in most studies, the 484
requirement for intact phospholipid has been increased growth, improved survival and reduced 485
malformations or deformities (Coutteau et al., 1997). Effects of nutritional components, including 486
dietary phospholipids, on skeletal development in fish larvae have been reviewed recently (Cahu 487
17
et al., 2003a). A limited number of studies have also looked at stress resistance and showed that 488
this could be increased by phospholipid supplementation (Takeuchi et al., 1992; Kanazawa, 489
1993; Weirich and Reigh, 2001). Few other physiological, metabolic, biochemical or molecular 490
parameters have been investigated (Fontagne et al., 2000; Koven, 2003; Zambonino-Infante and 491
Cahu, 2007; Hamza et al., 2008). 492
493
5.4 Range of species and developmental stages 494
A relatively defined quantitative requirement for dietary phospholipid has been determined in a 495
number of fish including the marine species red sea bream, Japanese flounder (Paralichthys 496
olivaceus), rock bream/knife jaw (Oplegnathus fasciatus), European sea bass (Dicentrarchus 497
labrax), striped jack (Pseudocaranx dentex), gilthead sea bream (Sparus aurata) and turbot, and 498
the freshwater species ayu, common carp, rainbow trout and white sturgeon (Acipenser 499
transmontanus) (see Table 1 for references). A semi-quantitative requirement has also been 500
determined in Atlantic salmon at the freshwater stage. The requirement for phospholipid has been 501
demonstrated at both larval and juvenile stages for ayu, Japanese flounder, rock bream/knife jaw 502
and European sea bass, in larvae for carp, red and gilthead sea bream, and in juveniles for striped 503
jack, turbot, rainbow trout, Atlantic salmon and white sturgeon. Based on studies comparing a 504
neutral lipid-rich commercial diet and a phospholipid-rich zooplankton diet, Olsen et al. (1991) 505
suggested that polar lipids may promote growth in juvenile Atlantic cod, but further work is 506
required to establish if there is a genuine requirement in this species. As mentioned previously, 507
few studies have been conducted but a requirement for phospholipid has not been conclusively 508
demonstrated in adults of any fish species (Olsen et al., 1999). 509
510
5.5 Quantitative requirements 511
Quantitative requirements have generally been reported in terms of phospholipid levels as a 512
percentage of the diet by weight. However, some data should be regarded as semi-quantitative 513
only, as the actual phospholipid content of the phospholipid preparations used in different 514
studies, which varies as described above, is not always taken into account and so the absolute 515
phospholipid level required may be lower than the value quoted in some cases. This also means 516
that it is not always appropriate to directly compare values in different trials using different 517
phospholipid preparations. Differences in the initial size of larvae or juveniles used, and the 518
durations of the feeding trials also contribute to the semi-quantitative nature of the data. Coutteau 519
et al. (1997) summarised the differences between the trials in all studies published up until the 520
18
date of that review. Acknowledging these caveats, values quoted for the quantitative 521
requirements of phospholipids for larval fish range from about 2 - 12 % of diet (Table 1). 522
Requirements were lower in the freshwater fish, carp (2 %) and ayu (3-5 %), and higher in the 523
marine fish red sea bream (5 %), rock bream/knife jaw (5-7 %) and Japanese flounder (7%), with 524
the highest reported value being for European sea bass at 12 % of diet. In juveniles, the values 525
ranged from around 1.5 - 7 % of diet, including 1.5 % for striped jack, 2-3 % for European sea 526
bass and around 4-6 % for Atlantic salmon, and 7 % for Japanese flounder. In Atlantic salmon, 527
studies showed that the phospholipid requirement was 4 - 6 % of diet in fish of initial size 180 528
mg, 4 % in fish of 1-1.7 g initial weight, whereas in fish of 7.5 g initial weight, no requirement 529
was observed. Similarly, in white sturgeon of 5-10 g, no requirement for dietary phospholipid 530
was detected. Therefore, the data appear to suggest a possible trend with quantitative 531
requirements decreasing with development, at least from larvae to small juveniles. No 532
requirement has been observed in fish (at least salmon and white sturgeon) of greater than 5 g 533
initial weight although this may be due to the short-term nature of the studies. Thus, in Atlantic 534
salmon cultured over a 24-month growth cycle low dietary phospholipid might induce growth 535
depression and other effects over this longer period of culture. 536
537
5.6 Qualitative requirements 538
There are few data available with which to compare the efficacy of different phospholipid classes 539
in satisfying phospholipid requirements. First of all, few phospholipid preparations have been 540
investigated, principally soybean lecithin (SL), egg lecithin (EL), but occasionally also some 541
marine phospholipids (Table 1). As mentioned above, all of these preparations are mixtures of 542
different phospholipid classes, but mainly PC, PE, PI and PS although the proportions vary. For 543
instance, SL can contain 50 - 86 % phospholipid, with typically around 20 – 25 % each of PC, PE 544
and PI. Therefore, it is not possible to draw any conclusions on the efficacy of individual 545
phospholipid classes in trials using commercial crude phospholipid preparations. However, some 546
studies have utilized semi-purified phospholipids, including soybean PC (80 - 98 % PC) or egg 547
PC (95 % PC), soybean PE (99 % PE) and soybean PI (Geurden et al., 1998a). Thus, in the few 548
studies in which comparisons are possible, both PC and PI were effective in meeting 549
phospholipid requirements in larval ayu whereas PE was less effective (Kanazawa, 1983; 550
Kanazawa et al., 1985). In Japanese flounder larvae, growth improved with PC, but not with PI or 551
PE (Kanazawa, 1993). When carp larvae were fed diets containing either purified phospholipid 552
classes (PC, PE, PS and PI), PC gave higher initial growth but induced deformities, whereas PI 553
19
gave optimal survival and minimal deformities (Geurden et al., 1998a). PE and PS gave 554
intermediate results, and hydrogenated PC or lyso-PC was not as effective as native PC. 555
Therefore, there are no clear patterns other than PC and PI may generally be more effective than 556
PE and PS, with PC perhaps being more important for growth and PI being more important for 557
normal development and prevention of malformations, at least in carp larvae. However, it is 558
possible that different phospholipid classes may play slightly different roles and thus have 559
different effects in different species as PI may have been more effective than PC in improving 560
growth in larval ayu (Kanazawa, 1983). 561
562
6. Mechanisms of requirement for intact phospholipids in larvae and juveniles 563
564
6.1 Improved diet qualities 565
It has been suggested that phospholipids may help to reduced leaching of water-soluble 566
micronutrients (minerals and vitamins) from semi-purified diets (see Coutteau et al., 1997). 567
Addition of phospholipid influenced the loss of dry matter in casein-based microdiets for post-568
larval shrimp (Camara, 1994). In a study investigating phospholipid requirements for European 569
sea bass and turbot using extruded diets with or without 2 % EL (69% pure), a diet water stability 570
test showed no effect of phospholipid on dietary fatty acid contents but micronutrients were not 571
investigated (Geurden et al., 1997b). This possible effect of dietary phospholipid requires further 572
study. 573
Although phospholipids contain oxidatively sensitive PUFAs, there is some evidence that they 574
can express an antioxidative effect on various oils and fats (Ishihara, 1997). Satoh and Ishihara 575
(1997) tested various compounds, representative of the major functional groups in phospholipids, 576
for antioxidant activity in a sardine oil system and found choline and ethanolamine strongly 577
inhibited increases in peroxide values in a sardine oil mixture during storage, whereas PA 578
derivatives and glycerol did not. Antioxidant effect was thus attributable not only to the side-579
chain amino groups but also to the cooperative effect of the hydroxy group in the side chain. The 580
precise mechanism of the antioxidant effect is unknown and various hypotheses include chelation 581
of transition metals, a synergistic effect with vitamin E, and hydroperoxide decomposing activity 582
(Ishihara, 1997). 583
PC enhanced feeding activity and diet ingestion rate of microdiets fed to sea bream larvae up 584
to day 30 post-hatch, which may suggest a role for dietary phospholipids as an age-dependent 585
feed attractant (Koven et al., 1998, 2001). An attractant property is likely to be dependent upon 586
20
the phospholipid head group as lecithin and certain L-amino acids had previously been tested for 587
attractant activity for aquatic organisms including weatherfish (Misgurunus anguillicaudatus) 588
and juvenile yellowtail (Seriola quinqueradiata) (Harada, 1987). The attraction activity of amino 589
acids and their derivatives was ascertained to largely depend on both alpha -carboxyl and alpha -590
amino groups, but especially the former. 591
592
6.2 Improved digestion 593
Phospholipids are surface-active agents, or surfactants, and the main industrial use of lecithins is 594
as lipid emulsifiers particularly in processed foods. Fish diets, of course, do not require the 595
presence of phospholipids as emulsifiers per se, but their presence in the formulation may 596
improve lipid emulsification and aid digestion in the intestine of the fish after consumption. 597
Improved lipid digestibility in salmon fed diets containing soybean lecithin has been attributed to 598
emulsification properties of the phospholipid (Hung et al., 1997), and other studies have shown 599
that dietary phospholipids increased digestibility in juvenile fish (Craig and Gatlin, 1997; Kasper 600
and Brown, 2003). However, supplementation with bile acids did not mimic the effect of dietary 601
phospholipid in promoting growth in larval ayu (Kanazawa, 1983). In a study in gilthead sea 602
bream larvae, a lecithin–supplemented diet substantially increased the uptake of labelled lipids 603
from the diet and this was initially suggested to be via improved emulsification (Koven et al., 604
1993). However, this result was not substantiated in subsequent studies on gilthead sea bream 605
fed with phospholipid-supplemented diets (Hadas et al., 2003). Phospholipid supplements also 606
aided the absorption of dietary neutral lipids in European sea bass post-larvae, but this was not 607
via a mechanism involving increased emulsification as diets containing other emulsifiers or lyso- 608
phospholipid, which has greater emulsification properties than phospholipid, did not replicate the 609
effect (Geurden et al., 1997c, 1998a). Therefore, there is little evidence to support a role for 610
dietary phospholipids as aids to digestion. 611
612
6.3 Provision of essential components 613
614
6.3.1 Fatty acids (energy and essential fatty acids) 615
Phospholipids can be an important source of energy (fatty acids) in fish, particularly during 616
embryonic and early larval development in species that produce phospholipid-rich eggs (Tocher, 617
1995). Larval fish at first feeding may be predisposed to digestion and metabolism of 618
phospholipids and the use of fatty acids from phospholipids for energy (Sargent et al., 1997). 619
21
Dietary lipid is also important as a source of essential fatty acids (EFA), and phospholipids tend 620
to be a richer source of EFA than neutral lipids such as triacylglycerols (Tocher, 1995). In 621
addition, phospholipids may be superior to neutral lipids as a source of EFA in larval fish due to 622
improved digestibility (Sargent et al., 1997, 1999b). Recently it was shown that phospholipids 623
were the more efficient mode of supply for dietary EPA and DHA to sea bass larvae (Gisbert et 624
al., 2005). However, in many experiments on phospholipid requirement, diets have been carefully 625
formulated to discriminate a phospholipid effect from a fatty acid effect with, for instance, diets 626
formulated to be EFA sufficient from neutral lipid alone (see Coutteau et al., 1997). In any case, 627
most of the studies on phospholipid requirement have been performed with soybean lecithin and 628
egg lecithin that are relatively rich in 18:2n-6 (and may have some 18:3n-3), but are deficient in 629
the n-3 HUFA, EPA and DHA, required in abundance by larval fish (Coutteau et al., 1997; 630
Sargent et al., 2002). In addition, further evidence that the beneficial effects of dietary 631
phospholipids are not due to the provision of EFA is that soybean lecithin, lacking n-3 HUFA is 632
as effective or more effective as lecithins from marine sources including fish eggs which are rich 633
in n-3HUFA (Kanazawa et al., 1981, 1983a; Geurden et al., 1995b). Therefore, there is good 634
evidence that the effects of dietary phospholipids on growth-promotion, survival and the 635
prevention of malformations are not due to the provision of EFA. Indeed, diets containing very 636
high levels of marine phospholipids and n-3HUFA actually induced skeletal malformations in sea 637
bass larvae (Villeneuve et al., 2005, 2006). 638
639
6.3.2 Phosphorous 640
Phosphorous is a nutritionally important mineral due to its requirement for growth, bone 641
mineralization, reproduction, nucleic acid synthesis, and energy metabolism (Lall, 2002). 642
Deficiency signs include reduced growth and skeletal deformities and quantitative requirements 643
have been determined for several fish species (Lall, 2002). As phosphate is low in most aquatic 644
environments (non-polluted), feed is the main source of phosphorous, mainly from meals or 645
premixes, with meat/bone meal > fish meal > plant meals. Bioavailability varies, but generally 646
the inorganic (calcium and potassium salts) and organic (phospholipids) forms found in fish 647
meals are more readily available to fish than the phytates (phytic acid salts) found in plant meals 648
(Lall, 2002). Many studies on phospholipid requirements have used casein- or soy protein-based 649
diets, but it is unlikely that this would be a problem with mineral premixes (Coutteau et al., 650
1997). In the only study to date, the effects of dietary phosphorus and phospholipid level on 651
growth and phosphorus deficiency signs were investigated in juvenile (1 g) Japanese flounder 652
22
(Uyan et al., 2007). The results showed there was no interaction between dietary phosphorous 653
and phospholipid suggesting that supply of phosphorous was not a mechanism for the growth 654
promoting effects of dietary phospholipids. 655
656
6.3.3 Choline or inositol 657
It is known that choline cannot be synthesised in animals (Lykidis, 2007), but there may be 658
evidence that some inositol can be synthesised in carp intestine and channel catfish (see Halver, 659
2002) and may not be required by juvenile sunshine bass (Deng et al., 2002). However, both 660
choline and inositol are regarded as vitamins for fish, as there are known deficiency signs for 661
both, and quantitative requirements for growth have been defined, at least for choline (Halver, 662
2002). Both choline and inositol are quite ubiquitous in feed ingredients including wheat germ, 663
fish and plant (bean) meals, but they are often also supplemented in vitamin premixes. In studies 664
with carp larvae, the growth-promoting and prevention of malformation effects of PC and PI 665
were not mimicked by choline and inositol (Geurden et al., 1995b). 666
667
6.4 Provision of intact phospholipid 668
Over twenty years ago, based on his own series of studies with larval ayu, red sea bream and 669
Japanese flounder, Kanazawa speculated that phospholipid biosynthesis itself may be limiting in 670
larval fish. He reasoned that, as larval fish require an abundance of phospholipid for new tissue 671
growth, “biosynthesis of phospholipids may not take place at a sufficiently fast rate to meet 672
phospholipid requirement” and “requirement for dietary phospholipids may diminish with 673
increasing age” (Kanazawa, 1985). However, it was another 10-15 years before there was a 674
sufficient body of evidence to support the hypothesis of limited phospholipid biosynthetic ability 675
in early life stages of fish. 676
Several studies showed that larval diets deficient in phospholipid could lead to the 677
accumulation of lipid vacuoles (droplets) in the intestinal enterocytes (Fontagné et al., 1998; 678
Olsen et al., 1999; Salhi et al., 1999; Liu et al., 2002). For example, intestinal steatosis (fat drop 679
accumulation) was induced in carp larvae fed phospholipid-deficient diets and PC and, to a lesser 680
extent, PI, prevented this (Fontagné et al., 1998). These studies led to the suggestion that intact 681
dietary phospholipids may be required for the efficient exportation of dietary lipids from the 682
enterocytes (Fontagné et al., 1998; Geurden et al., 1998b; Olsen et al., 1999; Salhi et al., 1999). 683
Values are means of duplicate analyses. BergaPur is a deoiled lecithin (98%) (Berg+Schmidt, Hamburg, Germany); DGDG, digalactosyldiacylglycerols; FeaM, feather meal; Hb, Heamoglobin; KB, kidney beans;
MGDG, monogalactosyldiacylglycerols; NFO, North Atlantic fish oil; PA, phosphatidic acid; PG, phosphatidylglycerol; RO, rapeseed oil; SF, sunflower; SFO, South American fish oil; SO, soybean oil.
Oils BindersMeals
45
1275
1276 1277
Table 4. Cholesterol content (mg/100g product) of
traditional feeds and alternative feed ingredients